The majority of nerve cell connections are chemical synapses. A neurotransmitter is released from the presynaptic terminal, typically in response to the arrival of an action potential in the neuron, and diffuses across the synaptic space to bind to membrane receptor proteins of the postsynaptic terminal. The binding of neurotransmitters to membrane receptors is coupled either to the generation of a permeability change in the postsynaptic cell or to metabolic changes.
Neurotransmitters produce different effects according to the type of receptor to which they bind. In general, those which produce effects that are rapid in onset and brief in duration bind receptors that act as ligand-gated ion channels, where binding almost instantly causes an ion flow across the membrane of the postsynaptic cell. Those neurotransmitters which act more like local chemical mediators bind to receptors that are coupled to intracellular enzymes, thereby producing effects that are slow in onset and more prolonged. These neurotransmitters alter the concentration of intracellular second messengers in the postsynaptic cell.
Four second messenger systems have been linked to neurotransmitter or hormone receptors and have been studied for their roles in the control of neuronal excitability. They are the adenylate cyclase/cyclic AMP-dependent protein kinase system, guanylate cyclase and cGMP-dependent protein kinase, the inositol trisphosphate/diacyl glycerol-protein kinase C system, and systems which are activated by calcium ions, such as the calcium/calmodulin-dependent protein kinase system. Thus, binding of a transmitter to a receptor may activate, for example, adenylate cyclase, thereby increasing the intracellular concentration of cAMP, which in turn activates protein kinases that phosphorylate specific proteins in the cells, such as those which form ion channels and thus alter the cells' electrical behavior. As with the ligand-gated ion channel transmitters, the effects can be either excitatory or inhibitory, and may affect the cell at many levels, including the pattern of gene expression. It is also believed that these chemical synapses, associated with second-messenger systems, may be involved in long-term changes that comprise the cellular basis of learning and memory.
The ligand-activated membrane receptors do not activate the second messenger systems directly, however, but via a membrane-bound protein, the GTP-binding protein (G protein), which binds GTP on the cytoplasmic surface of the cell membrane and thereby acts to couple adenylate cyclase to the membrane receptor. Neurotransmitter binding to the membrane receptor is believed to alter the conformation of the receptor protein to enable it to activate the G protein in the lipid bilayer, which then binds GTP at the cytoplasmic surface and produces a further change in the G protein to allow it to activate, e.g., an adenylate cyclase molecule to synthesize cAMP. When a ligand binds a receptor, an enzymatic cascade results as each receptor activates several molecules of G protein, which in turn activate more molecules of adenylate cyclase which convert an even larger number of ATPs to cAMP molecules, producing a substantial amplification from the initial event.
Glutamate, aspartate and their endogenous derivatives are believed to be the predominant excitatory neurotransmitters in the vertebrate central nervous system. (Krinjrvic, Phys. Rev. 54:418-540, 1974). Recently, glutamate has been described as playing a major, widespread role in the control of neuroendocrine neurons, possibly controlling not only the neuroendocrine system but other hypothalamic regions as well. Four major subclasses of glutamate receptors have been described but their characterization has until recently been limited to pharmacological and electrophysiological functional analyses. See generally, Hollman et al., Nature 342:643-648 (1989) and Sommer et al., Science 249:1580-1585 (1990). Three of the receptors, the quisqualate (QA/AMPA), kainate (KA), and N-methyl-D-aspartate (NMDA) receptors, are believed to be directly coupled to cation-specific ion channels and thus are classified as ligand-gated ionotropic receptors. The fourth glutamate receptor binds some of the agonists of the ionotropic receptors (quisqualate and glutamate, but not AMPA) but has no shared antagonists, and is coupled to G protein. Thus, this receptor, referred to as the G protein-coupled glutamate receptor, or Glu.sub.G R, is pharmacologically and functionally distinct from the other major glutamate receptors. This receptor has also been termed the metabotropic receptor.
Agonist binding to Glu.sub.G R has been shown to result in the activation of a number of second messenger systems, depending on the system studied. One of the best characterized is the quisqualate activation of phospholipase C through a G protein coupled interaction that leads to the stimulation of inositol phospholipid metabolism. This activity has been studied in systems that measure the accumulation of radiolabeled inositol monophosphate in response to stimulation by glutamate. The systems typically use brain slices from regions such as the hippocampus, striatum, cerebral cortex and hypothalamus (Nicoletti, et al., Proc. Natl. Acad. Sci. USA 83:1931-1935 (1986), and Nicoletti, et al., J. Neurochem. 46:40-46 (1986)) neuronal cultures derived from embryonic mouse and rat cerebellum, corpus striatum and cerebral cortex (Nicoletti et al., J. Neurosci. 6:1905-1911 (1986), Sladeczek et al., Nature 317:717-719 (1985), Dumuis, et al., Nature 347:182-184 (1990), and Drejer et al., J. Neurosci. 7:2910-2916 (1987)) and rat brain synaptosomes (Recasens et al., Eur. J. Pharm. 141: 87-93 (1987), and Recasens et al., Neurochem. Int. 13:463-467 (1988)). A major disadvantage of each of these model systems is the difficulty in analyzing the pharmacological and functional activities of Glu.sub.G R in an environment where other glutamate receptors and G protein-coupled receptors such as muscarinic and serotonin receptors are also present.
The Xenopus oocyte system has been used to identify Glu.sub.G R as a member of the family of G protein-coupled receptors. An endogenous inositol triphosphate second messenger-mediated pathway in the oocyte allows the detection of Glu.sub.G R after injection of total rat brain mRNA, in that the oocyte responds to ligand via the oocyte G protein-coupled PLC-mediated activation of a chloride channel that can be detected as a delayed, oscillatory current by voltage-clamp recording (Houamed et al., Nature 310:318-321 (1984); Gunderson et al., Proc. Royal Soc. B221:127-143 (1984); Dascal et al., Mol. Brain Res. 1:301-309 (1986); Verdoorn et al., Science 238:1114-1116 (1987); Sugiyama et al., Nature 325:531-533 (1987); Hirono et al., Neuros. Res. 6:106-114 (1988); Verdoorn and Dingledine, Mol. Pharmacol. 34:298-307 (1988); and Sugiyama et al., Neuron 3:129-132 (1989)). Injection of region-specific brain mRNA and of size fractionated mRNA have suggested that Glu.sub.G R may be a large mRNA (6-7 kb) and that it is enriched in the cerebellum (Fong et al., Synapse 2:657-665 (1988) and Horikoshi et al., Neurosci. Lett. 105:340-343 (1989)).
There remains considerable need in the art for isolated and purified Glu.sub.G R, as well as systems capable of expressing Glu.sub.G R separate from other neurotransmitter receptors. Further, it would be desirable to specifically identify the presence of Glu.sub.G R in cells and tissues, thereby avoiding the time-consuming, complex and nonspecific functional electrophysiological and pharmacological assays. It would also be desirable to screen and develop new agonists and/or antagonists specific for Glu.sub.G R, but to date this has not been practical. Quite surprisingly, the present invention fulfills these and other related needs.